CN111233880A - Preparation method of highly sensitive hypochlorite fluorescent probe with extremely low background fluorescence - Google Patents
Preparation method of highly sensitive hypochlorite fluorescent probe with extremely low background fluorescence Download PDFInfo
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- CN111233880A CN111233880A CN202010128712.7A CN202010128712A CN111233880A CN 111233880 A CN111233880 A CN 111233880A CN 202010128712 A CN202010128712 A CN 202010128712A CN 111233880 A CN111233880 A CN 111233880A
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- hypochlorite
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- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 title claims abstract description 145
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 78
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 239000000523 sample Substances 0.000 claims abstract description 56
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229910001868 water Inorganic materials 0.000 claims abstract description 18
- 241000252212 Danio rerio Species 0.000 claims abstract description 12
- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical compound NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 claims abstract description 6
- NRZXBDYODHLZBF-UHFFFAOYSA-N 63149-33-7 Chemical compound C1CCN2CCCC3=C2C1=CC(C=O)=C3O NRZXBDYODHLZBF-UHFFFAOYSA-N 0.000 claims abstract description 6
- 230000035945 sensitivity Effects 0.000 claims abstract description 6
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 5
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 54
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 30
- 238000001514 detection method Methods 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- KCDCNGXPPGQERR-UHFFFAOYSA-N coumarin 343 Chemical compound C1CCC2=C(OC(C(C(=O)O)=C3)=O)C3=CC3=C2N1CCC3 KCDCNGXPPGQERR-UHFFFAOYSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
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- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Chemical compound ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 claims description 10
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- 230000015572 biosynthetic process Effects 0.000 claims description 4
- QKIUAMUSENSFQQ-UHFFFAOYSA-N dimethylazanide Chemical compound C[N-]C QKIUAMUSENSFQQ-UHFFFAOYSA-N 0.000 claims description 4
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- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 claims 1
- 230000009471 action Effects 0.000 abstract description 6
- -1 coumarin 343 acyl chloride Chemical class 0.000 abstract 1
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- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 6
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
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- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
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- DQEUFPARIOFOAI-UHFFFAOYSA-N 2-propan-2-ylpropanedioic acid Chemical compound CC(C)C(C(O)=O)C(O)=O DQEUFPARIOFOAI-UHFFFAOYSA-N 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- WGKORGIPQUAKOI-UHFFFAOYSA-N 3-cyclopropyloxy-3-oxopropanoic acid Chemical compound OC(=O)CC(=O)OC1CC1 WGKORGIPQUAKOI-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
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- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 241000283070 Equus zebra Species 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 208000004852 Lung Injury Diseases 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
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- GXHFUVWIGNLZSC-UHFFFAOYSA-N meldrum's acid Chemical compound CC1(C)OC(=O)CC(=O)O1 GXHFUVWIGNLZSC-UHFFFAOYSA-N 0.000 description 1
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
- C07D491/16—Peri-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention belongs to the technical field of fluorescent probes, and particularly relates to a preparation method of a highly sensitive hypochlorite fluorescent probe with extremely low background fluorescence. The invention provides a preparation method for synthesizing a fluorescent probe by using 8-hydroxy julolidine-9-aldehyde as an initial raw material through the generated coumarin 343 acyl chloride and 2, 4-dinitrophenylhydrazine under the action of triethylamine; the fluorescent probe is a hypochlorite fluorescent probe with extremely low background fluorescence and high sensitivity, can selectively identify hypochlorite, and the fluorescence intensity of the hypochlorite fluorescent probe is gradually enhanced at 488nm along with the increase of the concentration of the hypochlorite; the probe has the functions of sensitivity and quick identification; the fluorescent probe prepared by the invention can be used for detecting the hypochlorite content in an actual water sample, detecting exogenous/endogenous hypochlorite in living cells and detecting hypochlorite in a zebra fish body.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to preparation of a highly sensitive hypochlorite fluorescent probe with extremely low background fluorescence and application of the highly sensitive hypochlorite fluorescent probe in detection of hypochlorite in actual water samples, living cells and zebra fish bodies.
Background
Hypochlorite (ClO)-) Is a common Reactive Oxygen Species (ROS) that has been widely used in our daily lives as disinfectants and household bleaches. Generally, the concentration is in the range of 10-104And mu.M. However, high levels of ClO exposure to water-The residue may cause eye, nose and stomach discomfort, thereby causing serious adverse effects on human health. On the other hand, hydrogen peroxide (H) is catalyzed by Myeloperoxidase (MPO)2O2) With chloride ion (Cl)-) Can produce ClO in vivo-. It plays an important role in the human immune system and in maintaining intracellular redox homeostasis. However, ClO in human body-An imbalance in concentration often leads to diseases associated with oxidative stress damage, such as cardiovascular disease, lung injury, rheumatoid arthritis, and the like. Therefore, there is an urgent need to construct a kit for detecting ClO in water and biological samples-An effective method of (1).
In recent years, various assays for ClO have been developed-The method of (3) includes spectrophotometry, colorimetry, chemiluminescence, electrochemical analysis, fluorescent probes, and the like. Fluorescent probes are of interest for their high selectivity, high sensitivity, ease of handling, and non-invasive nature for detection compared to other detection methods. Based on the strong oxidizing property of hypochlorite, fluorescent probes containing hydrazide, sulfur, hydrazone, oxime, p-methoxyphenol, carbon-carbon double bond and other recognition groups are designed. These fluorescent probes exhibit good analytical performance, but have the problem that the autofluorescence background of the probe molecules is high. In practical applications, the high background fluorescence signal often reduces the detection sensitivity and even results in false positive results. To obtain accurate and reliable detection, notThere is a constant need to develop ClO with very low background fluorescence-A fluorescent probe.
Disclosure of Invention
Based on the proposed requirements, the present inventors have made extensive literature research and intensive studies on this, and have provided a highly sensitive hypochlorite fluorescent probe with extremely low background fluorescence.
The technical scheme of the invention is that the high-sensitivity hypochlorite fluorescent probe with extremely low background fluorescence has the following molecular structure:
the specific synthesis is carried out according to the following steps:
(1) synthesis of intermediate compound coumarin 343: dissolving equivalent weight of 8-hydroxy julolidine-9-aldehyde and malonic acid cycloisopropyl ester (Meerkino acid) in ethanol, and dropwise adding 0.1 equivalent weight of piperidine and acetic acid into the solution; stirring at normal temperature, and heating and refluxing; cooling, pouring into ice water, performing suction filtration to obtain a filter cake, and performing silica gel column chromatography purification by using dichloromethane, methanol and acetic acid as eluent to obtain a yellow solid, namely coumarin 343;
(2) and (3) synthesizing a fluorescent probe molecule LH-1: dissolving the coumarin 343 obtained in the step (1) in dichloromethane, dropwise adding 20 equivalents of oxalyl chloride in ice bath, then dropwise adding N, N-dimethylamide (DMF for short), stirring for the first time at room temperature, removing the solvent by rotation, and drying in vacuum to obtain a dried product;
dissolving the obtained product in anhydrous dichloromethane, adding 2, 4-dinitrophenylhydrazine with equivalent weight in batches, dropwise adding anhydrous triethylamine with 20 equivalent weight, stirring for the second time at room temperature, removing the solvent by rotation to obtain a crude product, and purifying by silica gel column chromatography with an eluent to obtain a yellow solid which is a probe molecular compound LH-1; the hypochlorite fluorescent probe with extremely low background fluorescence and high sensitivity is prepared by the method.
Preferably, the 8-hydroxy julolidine-9-aldehyde, the cyclopropyl malonate, the ethanol, the piperidine and the acetic acid in the step (1) are used in an amount of 1.12 g: 0.74 g: 8mL of: 0.044 g: 0.1-0.2 mL.
Preferably, the volume ratio of the ethanol to the ice water in the step (1) is 8: 100.
preferably, the stirring time at normal temperature in the step (1) is 30-40 min, and the heating reflux time is 3 h.
Preferably, the volume ratio of the dichloromethane, the methanol and the acetic acid in the step (1) is 50: 1: 0.1.
Preferably, the coumarin 343, the dichloromethane, the oxalyl chloride and the N, N-dimethylamide in the step (2) are used in an amount of 100 mg: 10mL of: 0.6 mL: 0.1-0.2 mL.
Preferably, the time for the first stirring in the step (2) is 24 hours, and the time for vacuum drying is 2 to 3 hours.
Preferably, the amount of the coumarin 343, the anhydrous dichloromethane, the 2, 4-dinitrophenylhydrazine and the anhydrous triethylamine in the step (2) is 100 mg: 10mL of: 70 mg: 1 mL.
Preferably, the time for the second stirring in the step (2) is 48 hours; the eluting agent is dichloromethane.
The invention has the beneficial effects that:
(1) the highly sensitive hypochlorite fluorescent probe LH-1 with extremely low background fluorescence prepared by the invention contains two quenching groups: the N-N single bond in the hydrazide and the strong quenching group 2, 4-dinitrophenyl. The former free rotation can cause fluorescence quenching of the probe molecule, and the latter can also quench the fluorescence of the fluorophore through the d-PET mechanism. Therefore, probe LH-1 shows extremely low background fluorescence. Meanwhile, the d-PET mechanism is not influenced by the viscosity of an environmental medium; even in a solution with high viscosity, the probe will show low background fluorescence.
(2) The highly sensitive hypochlorite fluorescent probe LH-1 with extremely low background fluorescence prepared by the invention shows good spectral response performance to hypochlorite. The fluorescence spectrum properties of the probe were first investigated. The fluorescence intensity of the probe per se at 488nm is extremely low; when hypochlorite was added to the probe, fluorescence increased significantly at 488nm, and as the concentration of hypochlorite increased, the fluorescence intensity here increased greatly. Second, the purple of the probe was investigatedExternal absorption spectrum. The probe itself has strong absorption at 459nm, and after hypochlorite is added, the blue is shifted to 424 nm. Then, the selectivity of the probe was investigated. The probe pair of common active oxygen (H) is examined2O2、·OH、1O2、ClO-) And the fluorescence response of active Nitrogen (NO). The results show that hypochlorite (ClO) is the only target analyte-) Can cause a significant fluorescence intensity increase of the probe without significant fluorescence intensity changes of other analytes. Next, the response time of the research probe to different concentrations of hypochlorite is within 20 minutes. Finally, the effect of pH on fluorescent probe recognition of hypochlorite was investigated. When the pH is 6.0 to 11.0, the probe can be used for detecting hypochlorite. In addition, the influence of different detection systems on the recognition of hypochlorite by the probe is also studied. The probe response performance was best when the volume ratio of acetonitrile to PBS buffer was 2: 8.
(3) The highly sensitive hypochlorite fluorescent probe LH-1 with extremely low background fluorescence prepared by the invention is used for ClO with different concentrations in various actual water samples (taken from Yudaihe river, quiet lake and tap water in school of Jiangsu university)-The recovery rate is measured, and the result is satisfactory.
(4) The highly sensitive hypochlorite fluorescent probe LH-1 with extremely low background fluorescence prepared by the invention is successfully used for exogenous and endogenous hypochlorite fluorescent imaging visual detection in living cells.
(5) The novel fluorescent probe LH-1 prepared by the invention can be used for ClO in zebra fish-And (4) performing fluorescence imaging visual detection.
Drawings
FIG. 1 shows the synthetic route of fluorescent probe LH-1.
FIG. 2 (a) shows the fluorescent probe LH-1 (10. mu.M) versus different concentrations of ClO-(0-40. mu.M) fluorescence spectrum of the response. Illustration is shown: with and without ClO under a hand-held 365nm ultraviolet lamp-Photograph of probe LH-1 (10. mu.M) (40. mu.M); (b) is a fluorescent probe LH-1 (10. mu.M) with ClO at 488nm-(0-20. mu.M) concentration variation.
FIG. 3 shows fluorescent probes LH-1 (10. mu.M) and ClO-(40 μ M) UV-visible absorption spectra before and after action; wherein a is LH-1; b is LH-1+ ClO-。
FIG. 4 shows the fluorescence intensity response (. lamda.) of the reaction between the fluorescent probe LH-1 (10. mu.M) and each reactant (40. mu.M)ex＝415nm，λem488 nm); illustration is shown: fluorescent color of solution after reaction of LH-1 (10. mu.M) with 40. mu.M of each reactant (from left to right: blank, ClO)-、H2O2、·OH、1O2And NO).
FIG. 5 shows LH-1 (10. mu.M) with different concentrations of ClO-(0, 10, 20, 30, 40. mu.M) plot of fluorescence intensity as a function of time (. lamda.M)ex＝415nm，λem＝488nm)。
FIG. 6 shows addition of ClO-Graph of fluorescence intensity of LH-1 (10. mu.M) as a function of pH (lambda.) (40. mu.M) before (■) and after (●)ex＝415nm，λem＝488nm)。
FIG. 7 shows that probes LH-1 vs ClO are used in different volume ratios of acetonitrile to PBS-The response performance map of (1).
FIG. 8 shows the determination of cell viability by MTT method.
FIG. 9 shows that fluorescent probe LH-1 detects ClO in HeLa cells-A fluorescence image of (a); (a-e) HeLa cells were incubated with LH-1 (10. mu.M) for 30min, and then ClO was added to different concentrations-(0. mu.M, 5. mu.M, 10. mu.M, 15. mu.M and 30. mu.M) post-exposure images; (f-j) is a combined image of (a-e) the corresponding bright field image; (k) quantifying the mean fluorescence intensity in (a-e).
FIG. 10 shows the endogenous ClO in LH-1 and RAW 264.7 cells as fluorescent probes-Fluorescence images of the effects; wherein (a) RAW 264.7 cells are incubated with a fluorescent probe LH-1(10 mu M) for 30 min; (b) RAW 264.7 cells were pretreated with 4-aminobenzoic acid hydrazide (ABAH, 200 μ M) for 30min, and then incubated with LH-1(10 μ M) for 30 min; (c) stimulating RAW 264.7 cells with lipopolysaccharide (LPS, 20 μ g/mL) for 12h, and then incubating with LH-1(10 μ M) for 30 min; (d-f) is a merged image of a-c and its corresponding bright field image; (g) quantifying the mean fluorescence intensity in (a-c).
FIG. 11 shows that fluorescent probe LH-1 (10. mu.M) is used for detecting ClO in zebra fish bodies-Fluorescence imaging of the effect: (a-b) incubating zebrafish with LH-1 (10. mu.M) for 30 min; (c-d) LH-1 (10. mu.M) for zebrafishIncubate for 30min, then add ClO-(5. mu.M) further incubation for 30 min; (e-f) Zebra fish incubated with LH-1 (10. mu.M) for 30min, then with ClO-(10. mu.M) further incubation for 30 min; wherein (a, c, e) is a bright field, and (b, d, f) is a green fluorescent channel.
Detailed Description
The invention is further described below, but not limited to, with reference to the following figures and examples.
Example 1:
synthesizing a fluorescent probe, wherein the synthetic route is shown as figure 1;
(1) synthesis of intermediate compound coumarin 343: dissolving 8-hydroxy julolidine-9-aldehyde (1.12g,5.18mmol) and isopropylmalonate (Meldrum's acid) (0.74g,5.18mmol) in 8mL of ethanol in a 100mL round-bottomed flask, and dropwise adding piperidine (0.044g,0.52mmol) and 2 drops of acetic acid (0.1-0.2 mL) to the solution; stirring at normal temperature for 30min, and heating and refluxing for 3 h; cooling, pouring into 100ml of ice water, performing suction filtration, and performing silica gel column chromatography purification on a filter cake by using dichloromethane, methanol and acetic acid as eluent at the volume ratio of 50: 1: 0.1 to obtain a yellow solid (0.93g, the yield is 63%) which is coumarin 343;1HNMR(400MHz,CDCl3)δ(ppm):12.497(s,1H),8.506(s,1H),7.022(s,1H),3.412(q,J＝4.8Hz,4H),2.903(t,J＝6.4Hz,2H),2.806(t,J＝6.2Hz,2H),2.013(t,J＝5.6Hz,4H)；13CNMR(100MHz,DMSO-d6)δ(ppm):165.074,161.081,153.103,149.621,149.178,127.940,120.005,107.714,105.438,105.182,50.113,49.580,27.209,20.920,19.954.HRMS:m/z:calcd for C16H16NO4:286.10793[M+H]+,Found:286.10775.
(2) and (3) synthesizing a fluorescent probe molecule LH-1: dissolving coumarin 343(100mg,0.35mmol) in 10mL of dichloromethane in a 100mL round-bottom flask, dropwise adding oxalyl chloride (0.6mL,7.1mmol) in ice bath, then adding two drops of DMF (0.1-0.2 mL), stirring at room temperature for 24h, removing the solvent in a rotating manner, and drying in vacuum for 2-3 h to obtain a dried product;
the product obtained above was directly dissolved in 10mL of anhydrous dichloromethane, and an equivalent amount of 2, 4-dinitrophenylhydrazine (70mg,0.35mmol) was added in portions, and anhydrous triethylamine (1mL,7 mL) was added dropwise2mmol), stirring at room temperature for 48 h; and (3) removing the solvent by spinning, and purifying the crude product by using a silica gel column chromatography by using dichloromethane as an eluent to obtain a yellow solid (84mg, the yield is 51.6 percent), namely the probe molecular compound LH-1.1H NMR(400MHz,CDCl3)δ(ppm):10.721(s,1H),9.725(s,1H),9.163(d,J＝2.4Hz,1H),8.594(s,1H),8.297(dd,J1＝9.2Hz,J2＝2.4Hz,1H),7.057(s,1H),5.322(s,1H),3.412(m,4H),2.931(t,J＝6.4Hz,2H),2.811(t,J＝6Hz,2H),2.026(m,4H)；13C NMR(100MHz,CDCl3)δ(ppm):163.725,163.046,153.131,149.397,149.298,148.754,130.960,130.199,127.552,123.602,120.382,114.937,108.282,105.780,105.677,53.434,50.438,50.006,27.435,20.960,20.013；MS(ESI):m/z:calcd for C22H20N5O7:466.14[M+H]+,C22H19N5O7Na488.12[M+Na]+.Found:466.34and 488.41,respectively.
Example 2:
preparing a fluorescent probe and hypochlorite solution;
preparing a probe solution: an amount of the probe was weighed and dissolved in DMF to give a 1mM stock solution of the probe solution. Preparing a hypochlorite solution: a certain amount of sodium hypochlorite was dissolved in ultrapure water, transferred to a 100mL volumetric flask, and added to water to the scale line to obtain a hypochlorite stock solution with a concentration of 10 mM. Diluting the 10mM hypochlorite stock solution step by step to obtain 10-0.1 mM hypochlorite solution. 20 mu L of the probe stock solution and a certain volume of hypochlorite solution are added into a 2mL centrifuge tube, and a certain volume of acetonitrile-PBS mixed solution (the volume ratio is 2:8, PBS is phosphate buffer solution with 10mM pH being 7.40) is added into the centrifuge tube, and the total volume is 2mL, so that the fluorescence probe with the concentration of 10 mu M and hypochlorite solution with the concentration of 0-40 mu M are mixed to be detected.
Example 3:
fluorescent probe and ClO-Fluorescence spectroscopy of action;
FIG. 2 (a) shows the fluorescent probe LH-1 (10. mu.M) versus different concentrations of ClO-(0-40. mu.M) fluorescence spectrum of the response. The excitation spectrum used in the experiment is 415nm, and the emission wavelength range is 434 &650 nm. The excitation and emission slit widths were both 10nm, and the fluorescence measurement instrument used was a ThermoFisher Lumina spectrofluorometer. As can be seen from FIG. 2 (a), before hypochlorite is added, the fluorescence intensity of the probe itself is extremely low due to the double quenching effect of the free rotation of the N-N single bond in the hydrazide and the d-PET mechanism of the strong quenching group 2, 4-dinitrophenyl; after hypochlorite addition, the fluorescence intensity at 488nm increased greatly, and the fluorescence intensity gradually increased with the increase of hypochlorite concentration. When 4 equivalents of hypochlorite were added, the fluorescence intensity increased 739 times. Indicating that the addition of hypochlorite cleaved the hydrazide bond, releasing coumarin 343. At the same time, the solution also changed from non-fluorescent to blue-green fluorescent under 365nm hand-held uv lamp illumination (panel (a) inset). FIG. 2 (b) shows probe LH-1 (10. mu.M) with ClO at 488-(0 to 20. mu.M) concentration. The linear response range of the probe to hypochlorite is 0-20 mu M, and the lower detection limit is 10.5 nM. The probe is shown to be highly sensitive to the response of hypochlorite, and is expected to be used for detecting small change of the concentration of the hypochlorite in living cells.
Example 4:
fluorescent probe and ClO-Ultraviolet-visible spectrometry of the effect;
FIG. 3 is a diagram showing UV-VIS absorption spectra before and after the action of fluorescent probe with hypochlorite. The concentration of the fluorescent probe was 10. mu.M, and the hypochlorite concentration was 40. mu.M. The instrument used for UV-Vis absorption Spectroscopy was the Shimadzu UV-2600 UV-Vis spectrophotometer. As can be seen from FIG. 3, the probe itself has strong absorption at 459 nm; after hypochlorite addition, the absorption maximum wavelength blue shifts to 424 nm. Indicating that both double quenching groups are eliminated, and generating a new compound.
Example 5:
fluorescent probe pair ClO-The selectivity of the assay;
FIG. 4 shows fluorescent probe pairs ClO-Selection of assay. Fluorescent probes at a concentration of 10. mu.M were investigated for different active oxygen (ClO) concentrations of 40. mu.M-、H2O2、·OH、1O2) And the fluorescence response of active Nitrogen (NO). As can be seen from the figure, only Cl was addedO-Can cause a significant enhancement of the fluorescence spectrum of the fluorescent probe without significant change by other substances. Indicating probe pair ClO-Has better selectivity. FIG. 4 is an inset showing the change in fluorescence of the probe solution after addition of the substance. As can be seen from the figure, only ClO is present-The addition caused the probe solution to change from non-fluorescent to blue-green fluorescent. Indicating that the fluorescent probe can visually detect the ClO-。
Example 6:
fluorescent probes and ClO with different concentrations-Response time determination of action;
FIG. 5 shows fluorescent probes at a concentration of 10. mu.M and ClO at different concentrations (0, 10, 20, 30, 40. mu.M)-Time response graph of action. As can be seen from the figure, the fluorescent probe pairs ClO-The response time is within 20 minutes, and the requirement of monitoring an actual sample can be met. Meanwhile, after the fluorescence intensity reaches the maximum value, the fluorescence intensity does not change along with the time extension any more, and the fluorescence probe is proved to have good light stability.
Example 7:
ClO determination by solution pH vs. fluorescent probe-The influence of (a);
FIG. 6 shows addition of 40. mu.M ClO-Before (■) and after (●), the fluorescence intensity of the fluorescent probe at 10. mu.M changes with pH at 488nm, the pH range examined is 2.0-11.0, it can be seen that the probe itself is essentially non-fluorescent in this pH range, 40. mu.M ClO is added-Then, the fluorescence intensity of the probe is greatly enhanced within the range that the pH value is more than or equal to 6.0. Indicating that the fluorescent probe can be used in ClO under physiological conditions-Detection of (3).
Example 8:
determination of ClO by acetonitrile volume in detection system to fluorescent probe-The influence of (a);
FIG. 7 shows response of probe LH-1 to hypochlorite in different volume ratios of acetonitrile to PBS. As can be seen from the figure, when the volume ratio of acetonitrile is 20%, the probe is directed to ClO-The response performance is optimal.
Example 9:
the application of the fluorescent probe in the detection of an actual water sample;
the water samples selected by the invention respectively come from Yu dai river water, quiet lake water and Tap water in a working room in the university of Jiangsu. Before the experiment, the actual water sample is filtered by a 0.45 mu m microporous filter membrane. The detection system is acetonitrile and an actual water sample in a volume ratio of 2: 8. Adding a certain volume of probe stock solution and ClO into a detection system respectively-Stock solution to make the final concentration of probe 10. mu.M, ClO-The final concentrations were 0, 5, 10 and 20. mu.M.
TABLE 1 Probe vs. ClO in actual water sample-The concentration recovery was measured.
As can be seen from the table, all three water bodies contain small amount of ClO-(all below the limit). ClO in three water bodies-The recovery rates are all over 93 percent, which indicates that the fluorescent probe can be used for ClO in an actual water sample-And (4) carrying out quantitative detection.
Example 10:
evaluating the cytotoxicity of the fluorescent probe;
RAW 264.7 cells or HeLa cells were cultured at 1X 105The density of individual cells/well was seeded in each well of a 96-well plate and incubated for 24 hours. Fluorescent probe stock was diluted with cell culture medium containing 10% fetal bovine serum to final probe concentrations of 0, 10, 20, 30, 40 and 50 μ M. 100. mu.L of the above solution was added to each well of a 96-well plate and incubated for 12 hours. mu.L of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide solution (5mg ∙ mL) was then added to each well-1HEPES), at 37 ℃ for 4 hours. The residual solution was removed, 100 μ L of dimethyl sulfoxide was added to each well to dissolve formazan crystals, and absorbance at 490nm was collected using a Synergy | H5 microplate detector. FIG. 8 is a diagram showing the determination of cell viability by MTT method. As can be seen from the figure, the probe showed low cytotoxicity.
Example 11:
application of fluorescent probe to exogenous ClO in HeLa cells-Detecting fluorescence imaging;
HeLa cells at 4X 104Per mL-1The cells of (2) were seeded on a 24-well plate, and a medium containing 10% FBS and 1% antibiotic (penicillin/streptomycin, 100U/mL) was added and incubated at 37 ℃ for 24 hours in a carbon dioxide incubator. The cells were divided into five groups, each of which was added with a certain volume of fluorescent probe stock solution to a final concentration of 10 μ M, and incubated for 30 minutes. The solution was then washed 3 times with PBS buffer to remove the fluorescent probes. Adding culture medium, adding ClO of certain volume into each group-The final concentrations of the stock solutions were 0, 5, 10, 15 and 30. mu.M, respectively. After further 30 minutes at 37 ℃ the plates were washed 3 times with PBS. Finally, the images were imaged by means of an inverted fluorescence microscope with a 20-fold objective on a Zeiss GmbH 37081 type inverted fluorescence microscope. The results are shown in FIG. 9. As can be seen from the figure, the fluorescence intensity gradually increases with the increase of the fluorescence intensity of hypochlorite, which indicates that the fluorescent probe can be used for the fluorescence imaging detection of exogenous hypochlorite with different concentrations.
Example 12:
fluorescent probe applied to endogenous ClO in RAW 264.7-Detecting fluorescence imaging;
RAW 264.7 cells at 6X 104Per mL-1The cells of (2) were seeded on a 24-well plate, and a medium containing 10% FBS and 1% antibiotic (penicillin/streptomycin, 100U/mL) was added and incubated at 37 ℃ for 24 hours in a carbon dioxide incubator. Cells were divided into three groups. The first group was the incubation of RAW 264.7 macrophages with fluorescent probes at a concentration of 10 μ M for 30 minutes. The second group was prepared by incubating RAW 264.7 macrophages with 200. mu.M of 4-aminobenzoic acid hydrazide (ABAH for short) for 30 minutes, followed by incubation with 10. mu.M of fluorescent probe for 30 minutes. In the third group, RAW 264.7 macrophages were stimulated with lipopolysaccharide (LPS for short, 20. mu.g/mL) for 12h, and then incubated with 10. mu.M fluorescent probe for 30 min. All cells above were washed 3 times with PBS buffer before imaging, and medium was added. Finally, the images were imaged by means of an inverted fluorescence microscope with a 20-fold objective on a Zeiss GmbH 37081 type inverted fluorescence microscope. The results are shown in FIG. 10. Can be seen from the figureAfter the RAW 264.7 cells are incubated by the fluorescent probe, weak fluorescence exists; fluorescence disappeared after addition of the Myeloperoxidase (MPO) inhibitor ABAH. Indicating that RAW 264.7 cells contain a small amount of endogenous hypochlorite. It is known that addition of LPS induces more hypochlorite in RAW 264.7 cells. The results in fig. 10c show a significant increase in fluorescence upon LPS stimulation. The results show that the fluorescent probe can be used for fluorescence imaging detection of endogenous hypochlorite in RAW 264.7 cells.
Example 13:
fluorescent probe applied to ClO in zebra fish body-Detecting fluorescence imaging;
incubating 3-day-old zebra fish with 10 μ M fluorescent probe in light incubator for 30min, and culturing with zebra fish embryo culture solution (0.1% NaCl, 0.003% KCl, 0.004% CaCl)2·H2O and 0.008% MgSO4) And washing to remove the remaining fluorescent probe. At the same time, other two groups of zebrafish were incubated with 10. mu.M fluorescent probe for 30 minutes, and then 5. mu.M ClO and 10. mu.M ClO were added-Incubate for 30 minutes and wash 3 times with culture. Fluorescence imaging was observed on a SZX2-ILLT type stereofluorescence microscope. The results are shown in FIG. 11. As can be seen from the figure, the addition of hypochlorite causes the zebra fish body to show green fluorescence; the greater the hypochlorite concentration, the stronger the fluorescence intensity. The fluorescent probe can be used for detecting hypochlorite in the zebra fish body.
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (10)
1. A high-sensitivity hypochlorite fluorescent probe with extremely low background fluorescence is characterized in that the molecular structural formula is as follows:
2. a preparation method of a high-sensitivity hypochlorite fluorescent probe with extremely low background fluorescence is characterized by comprising the following specific steps:
(1) synthesis of intermediate compound coumarin 343: dissolving equivalent weight of 8-hydroxy julolidine-9-aldehyde and malonic acid cycloisopropyl ester (Meerkino acid) in ethanol, and dropwise adding 0.1 equivalent weight of piperidine and acetic acid into the solution; stirring at normal temperature, and heating and refluxing; cooling, pouring into ice water, performing suction filtration to obtain a filter cake, and performing silica gel column chromatography purification by using dichloromethane, methanol and acetic acid as eluent to obtain a yellow solid, namely coumarin 343;
(2) and (3) synthesizing a fluorescent probe molecule LH-1: dissolving the coumarin 343 obtained in the step (1) in dichloromethane, dropwise adding 20 equivalents of oxalyl chloride under ice bath, then dropwise adding N, N-dimethylamide, stirring for the first time at room temperature, rotationally removing the solvent, and drying in vacuum to obtain a dried product;
dissolving the obtained product in anhydrous dichloromethane, adding 2, 4-dinitrophenylhydrazine with equivalent weight in batches, dropwise adding anhydrous triethylamine with 20 equivalent weight, stirring for the second time at room temperature, removing the solvent by rotation to obtain a crude product, and purifying by silica gel column chromatography with an eluent to obtain a yellow solid which is a probe molecular compound LH-1; the hypochlorite fluorescent probe with extremely low background fluorescence and high sensitivity is prepared by the method.
3. The method for preparing a highly sensitive hypochlorite fluorescence probe with very low background fluorescence as claimed in claim 2, wherein the 8-hydroxy julolidine-9-aldehyde, the cycloisopropyl malonate, the ethanol, the piperidine and the acetic acid in step (1) are used in an amount of 1.12 g: 0.74 g: 8mL of: 0.044 g: 0.1-0.2 mL.
4. The method for preparing a highly sensitive hypochlorite fluorescent probe with extremely low background fluorescence as claimed in claim 2, wherein the volume ratio of ethanol to ice water in step (1) is 8: 100, respectively; the stirring time at normal temperature is 30-40 min, and the heating reflux time is 3 h.
5. The method for preparing a highly sensitive hypochlorite fluorescence probe with very low background fluorescence as claimed in claim 2, wherein the volume ratio of dichloromethane, methanol and acetic acid in step (1) is 50: 1: 0.1.
6. The method for preparing a highly sensitive hypochlorite fluorescence probe with extremely low background fluorescence as claimed in claim 2, wherein the dosage relationship of the coumarin 343, the dichloromethane, the oxalyl chloride and the N, N-dimethyl amide in the step (2) is 100 mg: 10mL of: 0.6 mL: 0.1-0.2 mL.
7. The method for preparing a highly sensitive hypochlorite fluorescence probe with very low background fluorescence as claimed in claim 2, wherein the time for the first stirring in step (2) is 24h, and the time for vacuum drying is 2-3 h.
8. The method for preparing a highly sensitive hypochlorite fluorescent probe with extremely low background fluorescence as claimed in claim 2, wherein the dosage relationship of the coumarin 343, the anhydrous dichloromethane, the 2, 4-dinitrophenylhydrazine and the anhydrous triethylamine in the step (2) is 100 mg: 10mL of: 70 mg: 1 mL.
9. The method for preparing a highly sensitive hypochlorite fluorescence probe with very low background fluorescence as claimed in claim 2, wherein the time of the second stirring in step (2) is 48 h; the eluting agent is dichloromethane.
10. The highly sensitive hypochlorite fluorescence probe with extremely low background fluorescence as claimed in claim 1 is applied to detection of hypochlorite in water samples, living cells and zebra fish bodies.
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